Identification of human progenitors of exhausted CD8+ T cells associated with elevated IFN-γ response in early phase of viral infection.

T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of individuals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections.

Haematopoietic Stem Cell Transplantation Results in Extensive Remodelling of the Clonal T Cell Repertoire in Multiple Sclerosis.

Autologous haematopoietic stem cell transplantation (AHSCT) is a vital therapeutic option for patients with highly active multiple sclerosis (MS). Rates of remission suggest AHSCT is the most effective form of immunotherapy in controlling the disease. Despite an evolving understanding of the biology of immune reconstitution following AHSCT, the mechanism by which AHSCT enables sustained disease remission beyond the period of lymphopenia remains to be elucidated. Auto-reactive T cells are considered central to MS pathogenesis. Here, we analyse T cell reconstitution for 36 months following AHSCT in a cohort of highly active MS patients. Through longitudinal analysis of sorted naïve and memory T cell clones, we establish that AHSCT induces profound changes in the dominant T cell landscape of both CD4+ and CD8+ memory T cell clones. Lymphopenia induced homeostatic proliferation is followed by clonal attrition; with only 19% of dominant CD4 (p <0.025) and 13% of dominant CD8 (p <0.005) clones from the pre-transplant repertoire detected at 36 months. Recovery of a thymically-derived CD4 naïve T cell repertoire occurs at 12 months and is ongoing at 36 months, however diversity of the naïve populations is not increased from baseline suggesting the principal mechanism of durable remission from MS after AHSCT relates to depletion of putative auto-reactive clones. In a cohort of MS patients expressing the MS risk allele HLA DRB1*15:01, public clones are probed as potential biomarkers of disease. AHSCT appears to induce sustained periods of disease remission with dynamic changes in the clonal T cell repertoire out to 36 months post-transplant.

Prophylactic antigen-specific T-cells targeting seven viral and fungal pathogens after allogeneic haemopoietic stem cell transplant.

OBJECTIVES: Adoptive immunotherapy using donor-derived antigen-specific T-cells can prevent and treat infection after allogeneic haemopoietic stem cell transplant (HSCT). METHODS: We treated 11 patients with a prophylactic infusion of 2 × 107 cells per square metre donor-derived T-cells targeting seven infections (six viral and one fungal) following HSCT. Targeted pathogens were cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, varicella zoster virus, influenza, BK virus (BKV) and Aspergillus fumigatus. RESULTS: T-cell products were successfully generated in all patients with 10 products responsive to 6 or 7 infections. T-cell infusions were associated with increases in antigen-experienced activated CD8+ T-cells by day 30. CMV, EBV and BKV reactivation occurred in the majority of patients and was well controlled except where glucocorticoids were administered soon after T-cell infusion. Three patients in that circumstance developed CMV tissue infection. No patient required treatment for invasive fungal infection. The most common CMV and EBV TCR clonotypes in the infusion product became the most common clonotypes seen at day 30 post-T-cell infusion. Donors and their recipients were recruited to the study prior to transplant. Grade III/IV graft-versus-host disease developed in four patients. At a median follow-up of 390 days post-transplant, six patients had died, 5 of relapse, and 1 of multi-organ failure. Infection did not contribute to death in any patient. CONCLUSION: Rapid reconstitution of immunity to a broad range of viral and fungal infections can be achieved using a multi-pathogen-specific T-cell product. The development of GVHD after T-cell infusion suggests that infection-specific T-cell therapy after allogeneic stem cell transplant should be combined with other strategies to reduce graft-versus-host disease.

Ex vivo enrichment of PRAME antigen-specific T cells for adoptive immunotherapy using CD137 activation marker selection.

OBJECTIVE: Adoptive immunotherapy with ex vivo expanded tumor-specific T cells has potential as anticancer therapy. Preferentially expressed antigen in melanoma (PRAME) is an attractive target overexpressed in several cancers including melanoma and acute myeloid leukaemia (AML), with low expression in normal tissue outside the gonads. We developed a GMP-compliant manufacturing method for PRAME-specific T cells from healthy donors for adoptive immunotherapy. METHODS: Mononuclear cells were pulsed with PRAME 15-mer overlapping peptide mix. After 16 h, activated cells expressing CD137 were isolated with immunomagnetic beads and cocultured with irradiated CD137neg fraction in medium supplemented with interleukin (IL)-2, IL-7 and IL-15. Cultured T cells were restimulated with antigen-pulsed autologous cells after 10 days. Cellular phenotype and cytokine response following antigen re-exposure were assessed with flow cytometry, enzyme-linked immunospot (ELISPOT) and supernatant cytokine detection. Detailed phenotypic and functional analysis with mass cytometry and T-cell receptor (TCR) beta clonality studies were performed on selected cultures. RESULTS: PRAME-stimulated cultures (n = 10) had mean expansion of 2500-fold at day 18. Mean CD3+ percentage was 96% with CD4:CD8 ratio of 4:1. Re-exposure to PRAME peptide mixture showed enrichment of CD4 cells expressing interferon (IFN)-γ (mean: 12.2%) and TNF-α (mean: 19.7%). Central and effector memory cells were 23% and 72%, respectively, with 24% T cells expressing PD1. Mass cytometry showed predominance of Th1 phenotype (CXCR3+/CCR4neg/CCR6neg/Tbet+, mean: 73%) and cytokine production including IL-2, IL-4, IL-8, IL-13 and GM-CSF (2%, 6%, 8%, 4% and 11%, respectively). CONCLUSION: PRAME-specific T cells for adoptive immunotherapy were enriched from healthy donor mononuclear cells. The products were oligoclonal, exhibited Th1 phenotype and produced multiple cytokines.

Rapidly expanded partially HLA DRB1-matched fungus-specific T cells mediate in vitro and in vivo antifungal activity.

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice-compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen-matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

Could cannabis liberalisation lead to wider changes in drug policies and outcomes?

Cannabis policies are changing in some countries. This may have consequences that extend beyond cannabis-specific outcomes, such as an impact on the consumption patterns of other substances. Changes in cannabis policies may also influence policy responses to other drugs, as countries re-assess the balance between law enforcement and public health objectives. If this happens, it could have important health and social consequences, especially in those countries where a 'war on drugs' policy perspective has inhibited investment in evidence based responses in areas such as treatment and harm reduction. The burden of disease associated with opioid use for example is large and this is an area in which treatment and harm reduction have been shown to deliver benefits. Thus if the changes in cannabis policies result in a greater willingness to invest in effective interventions for other drugs, the potential net health gains could be considerable. On the other hand, if cannabis policy changes are associated with an increase in health risk behaviours, such as driving under the influence or increased use of harmful substances such as tobacco, then significant increased health costs could result. To date most attention has been focused on recent cannabis sales liberalisation in the Americas, but experiences from elsewhere are also informative. In Europe, for example, moves towards decriminalisation of drug possession are resulting in lower rates of incarceration and arguably have reduced barriers to treatment uptake. Robust monitoring and assessment of the impact of these different policy changes is crucial to evaluating and understanding their results. It is important that such monitoring is international in scope, is not limited to issues around the use of cannabis only, and considers the interactions that may exist between cannabis policies and the approaches taken to other substances.

Controlling new drugs under marketing regulations.

AIMS: The rapid emergence of myriad substances openly marketed as 'legal highs' is straining traditional drug control systems which require time and basic scientific data on harms to react, presenting governments with the dilemma of no response or a disproportionate response. Some countries have side-stepped this using novel policy and legislative approaches. Should other countries consider them? METHODS: We review the different laws invoked to stop the open sale of new psychoactive substances, focusing on the European Union (EU). RESULTS: Some countries have designed new catch-all control systems, or faster systems to classify substances as drugs. Others have enforced consumer safety or medicines legislation to stop the open sale of these products. The latter originate from harmonization of the internal market of the EU. Rigorous, objective evaluation is required, but first results suggest that these have been effective, while avoiding criminalization of users. CONCLUSIONS: Every EU country should have existing laws for protecting public health that can be applied swiftly yet proportionately to new drugs appearing on the open market with minimum political involvement. It seems the key is the speed, not the weight, of response. Given support for their enforcement mechanisms, these systems might be as effective and more efficient than the old ones.

Khat use and monitoring drug use in Europe: the current situation and issues for the future.

AIM OF THE STUDY: To review the information available on the use of khat (Catha edulis) in the EU, and to assess the future use of this drug and related substances. MATERIAL AND METHODS: Khat is not controlled by international law and it has not been systematically included in the list of illicit drugs monitored in the EU. The current principal source of information on khat use in Europe is the early-warning system set up to monitor new and emerging drugs. Further information was obtained from official national reports to the EMCDDA and from the scientific literature. RESULTS: Across Europe, the use of khat is low. Khat use is limited to countries with immigrant communities from countries where khat use is common (such as Ethiopia, Somalia and Kenya). Information on the prevalence of khat use in the general population is scarce. Data on seizures provide an insight on the situation, though these may be difficult to interpret. The most recent estimates suggest that Europe accounts for about 40% of the khat seized worldwide. CONCLUSION: The shortage of data on the use and patterns of use of khat in Europe does not allow an evaluation of the needs for health and social interventions in communities in which the drug is used. But seizures of the plant are increasing in the EU, and more synthetic derivatives of the pharmacologically active ingredients of the plant (cathine and cathinone) are appearing on the market. Some of these, like mephedrone, have significant potential for future diffusion, and are likely to play a greater role on the European drug scene of the future.

New for old? Risperidone long-acting injection in older patients.

OBJECTIVE: To describe six older patients who were treated with risperidone long-acting injection, and to discuss the role of this medication in a psychogeriatric setting. METHOD: Case series. RESULTS: Of the six patients trialled on risperidone long-acting injection, two remain on this treatment. Of the other four, three required a change of treatment due to a combination of adverse side-effects and non-response, and one due to adverse side-effects alone. CONCLUSIONS: Risperidone long-acting injection may have a role to play in the management of older patients with psychotic disorders but further research is required.